Kazuhiko Iwahashi

Isolation of Borna Disease Virus from Human Brain Tissue

ABSTRACT Serological and molecular epidemiological studies indicate that Borna disease virus (BDV) can infect humans and is possibly associated with certain neuropsychiatric disorders. We examined brain tissue collected at autopsy from four schizophrenic patients and two healthy controls for the presence of BDV markers in 12 different brain regions. BDV RNA and antigen was detected in four brain regions of a BDV-seropositive schizophrenic patient (P2) with a very recent (2 years) onset of disease. BDV markers exhibited a regionally localized distribution. BDV RNA was found in newborn Mongolian gerbils intracranially inoculated with homogenates from BDV-positive brain regions of P2. Human oligodendroglia (OL) cells inoculated with brain homogenates from BDV-positive gerbils allowed propagation and isolation of BDVHuP2br, a human brain-derived BDV. Virus isolation was also possible by transfection of Vero cells with ribonucleoprotein complexes prepared from BDV-positive human and gerbil brain tissues. BDVHuP2br was genetically closely related to but distinct from previously reported human- and animal-derived BDV sequences.

Studies of the immunohistochemical and biochemical localization of the cytochrome P-450scc-linked monooxygenase system in the adult rat brain

Immunohistochemical and biochemical studies were performed on the brains of adult female and male rats using a specific antibody against bovine adrenocortical cytochrome P-450scc. The results showed that in both male and female rats, the myelinated regions of the white matter are selectively immunostained throughout the brain and that even in rats pretreated with colchicine, there is never positive staining of neuronal cell bodies and their dendrites in any brain region. Western immunoblotting with the P-450scc antibody and enzymatic assays revealed that P-450scc and cholesterol side-chain cleavage activity were present in a homogenate derived from the cortical white matter, but not detectable in that from the cerebral cortex. Furthermore, quantitation of the P-450scc protein in the immunoblots indicated that the concentration of P-450scc in the cortical white matter of both female and male rat brains is approx. 3-4 pmol per mg tissue protein. Thus it could be concluded that in the adult rat brain, P-450scc and cholesterol side-chain cleavage activity are selectively localized only in the myelinated region of the white matter.

Linkage disequilibrium and association with methamphetamine dependence/psychosis of μ -opioid receptor gene polymorphisms

Several studies indicate that the μ-opioid receptor plays a role in addiction not only to opiate drugs but also to alcohol and non-opiate addictive drugs. Our studies aim to reveal the associations between gene polymorphisms and methamphetamine (MAP) dependence/psychosis. We newly identified several polymorphisms and four substantial linkage disequilibrium (LD) blocks in the μ-opioid receptor (OPRM1) gene. We found significant differences in both genotype and allele frequencies of the single-nucleotide polymorphism (SNP) IVS2+G691C between control (n=232) and MAP-dependent/psychotic patients (n=128). There was also a significant association between IVS2+G691C and patients with transient psychosis. These results suggest that the OPRM1 gene variations may be a factor in development and prognosis of MAP psychosis.

Clinical investigation of the relationship between Borna disease virus (BDV) infection and schizophrenia in 67 patients in Japan

The relationship between Borna disease virus (BDV) infection and schizophrenia in the clinical time course was investigated. By nested reverse‐transcribed polymerase chain reaction (RT‐PCR) and Western blotting, BDV‐specific RNA and anti‐BDV antibodies were examined in the EDTA‐treated blood from 67 schizophrenic patients (according to DSM‐III‐R) in Japan. A significantly higher proportion (45%) of anti‐BDV antibody and/ or BDV RNA carriers were found among these 67 schizophrenic patients than in 26 controls (0%). There were no apparent associations of BDV infection with age, age at onset, period of hospitalization, accompanying somatic diseases, a past history of tuberculosis, a history of transfusion, a family history, or doses of psychotropic drugs. It is possible that, at least, BDV infection in schizophrenic patients may not be a nosocomial (hospital‐acquired) infection, although the route of BDV infection in humans remains unidentified. More studies on the relationship between BDV infection and clinical psychosomatic features should be performed in order to elucidate the pathogenesis of schizophrenia.

Detection of anti-Borna Disease Virus (BDV) antibodies from patients with schizophrenia and mood disorders in Japan

The relationship between infection with the Borna Disease Virus (BDV) and the clinical symptoms of schizophrenia and mood disorders (DMS-IV) was investigated. Western blotting techniques were used to examine anti-p10-BDV antibodies in serum from 32 patients with schizophrenia and 33 patients with mood disorders in Japan. The results showed that 1 out of 25 controls (4.0%), 7 out of 32 patients with schizophrenia (21.9%) and 9 out of 33 patients with mood disorders (27.3%) were positive for anti-BDV-p10 antibodies. Compared with levels of anti-BDV-p10 antibodies in controls, the production of anti-BDV-p10 antibodies failed to show a statistically significant relationship with schizophrenia but did show a significant relationship with mood disorder. The subgroup of schizophrenia patients with positive syndromes had a non-significantly higher frequency of anti-BDV-p10 antibodies than the subgroup of patients with negative syndromes. Similarly, the production of anti-BDV-p10 antibodies was non-significantly higher among patients with the unipolar subtype of mood disorder than in those with the bipolar subtype.

Tissue‐specific alternative splicing of mouse brain type ryanodine receptor/calcium release channel mRNA

We detected alternative splicing of the mouse brain type ryanodine receptor (RyR3) mRNA. The splicing variant was located in the transmembrane segment. The non‐splicing type (RyR3‐II) included a stretch of 341 bp, and that of the 13th codon was stop codon TAA. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis shows that RyR3‐II mRNA was expressed in various peripheral tissues and brain at all developmental stages. However, interestingly, the splicing type (RyR3‐I) mRNA was detected only in the cerebrum. These findings suggest that the splicing variants RyR3‐I and RyR3‐II may generate functional differences of RyR3 in a tissue‐specific manner.

Whole Deletion of CYP2A6 Gene (CYP2A6*4C) and Smoking Behavior

The relationship between nicotine metabolism of CYP2A6 and the smoking behavior in a Japanese population was investigated. The CYP2A6 genotypes were determined by the PCR method. There was a significant difference in the frequency of the CYP2A6*4C allele, which is a whole deleted allele for the human CYP2A6 gene, between smokers and nonsmokers. The frequency of the CYP2A6*4C allele was higher in nonsmokers than in smokers, whereas the frequency of CYP2A6*1A/*1B heterozygotes with a higher activity of nicotine metabolism was lower. In this study it was suggested that the CYP2A6*4C allele may prevent the carrier from smoking and the CYP2A6*1A/*1B heterozygote may be at risk for developing smoking behavior.

Acetaldehyde adducts in the brain of alcoholics

Acetaldehyde binds to some proteins and becomes a Schiff base. It is assumed that after the consumption of ethanol the acetaldehyde binds to the proteins to form adducts, and such acetaldehyde adducts are associated with organ diseases. We investigated the detection of acetaldehyde adducts in the brain region of a human alcoholics. Brain samples collected from an alcoholic autopsied case were used. Determination of acetaldehyde adducts was performed using a fluorescence immmunohistochemical staining method with antibodies against acetaldehyde adducts. We demonstrated acetaldehyde adducts in the frontal cortex and the midbrain of an alcoholics. Our studies showed that an acetaldehyde adduct was produced in the brain of alcoholics.

The drug-drug interaction effects of haloperidol on plasma carbamazepine levels

The metabolic interaction between carbamazepine (CBZ) and haloperidol (HP) was studied in Japanese schizophrenic patients treated with HP but not with CBZ and with both CBZ and HP. The serum CBZ concentrations in patients treated without HP were significantly decreased (p < 0.05), on average approximately 40%, as compared to those in patients treated with both CBZ and HP, whereas the serum HP concentrations in patients treated with both HP and CBZ were significantly decreased (p < 0.05), as compared to those in patients treated with HP but not with CBZ. The effect of HP, which prevents the serum CBZ level from decreasing, was shown in this study.

Evidence that variation in the peripheral benzodiazepine receptor (PBR) gene influences susceptibility to panic disorder

Panic disorder (PD) is the repeated sudden occurrence of panic attacks, episodes characterized by psychological symptoms. Peripheral benzodiazepine receptor (PBR) is closely associated with personality traits for anxiety tolerance, and that it holds promise as a biological marker of stressful conditions. We have performed association analyses using the polymorphism to determine the PBR in PD. We screened the subjects for sequence variations within the 5′ region, the coding region (exons 2–4), and the 3′ noncoding region. One novel missense variant in exon 4, derived from the nucleotide transition in codon 162 (CGT → CAT:485G > A) resulting in an arginine‐to‐histidine (Arg → His) change, was detected in these subjects. The 485G > polymorphism of the PBR gene was analyzed in 91 PD patients and 178 controls. The genotypic and allelic analyses of the 485G > A revealed significant differences between the panic patients and the comparison subjects (P = 0.021 and 0.014, respectively). The present study provides new and important evidence that variation in the PBR gene influences susceptibility to PD.